Proteins required for the binding of mitochondrial ATPase to the mitochondrial inner membrane

1. Isolated F₁ (mitochondrial ATPase) binds to urea-treated submitochondrial particles suspended in sucrose/Tris/EDTA with a dissociation constant of 0.1 μM.

2. About one-third of the F₁ and the oligomycin-sensitivity conferring protein (OSCP) are lost during preparation of submitochondrial particles prepared at high pH (A particles). None is lost from particles treated with trypsin (T particles).

3. After further treatment with alkali of urea-treated particles, binding of F₁ requires the addition of OSCP. Maximum binding is reached when both OSCP and Fc₂ are added. The concentration of F₁-binding sites in the presence of both OSCP and Fc₂ is about the same as that in TU particles.

4. After further extraction with silicotungstate of urea- and alkali-treated particles, OSCP no longer induces binding of F₁, unless Fc₂ is also present. Fc₂ induces binding in the absence of OSCP but with a lower binding constant and, in contrast to results under all the other conditions studied in this paper, the ATPase activity is oligomycin insensitive.

5. It is tentatively concluded that OSCP is the binding site for F₁ and Fc₂ is the binding site for OSCP.     

The mechanism of inhibition by oligomycin of oxidative phosphorylation in mitochondria

1. The concentration of specific oligomycin-binding sites in rat-liver mitochondria is 0.12 nmole/mg protein, whereas at least 10-times more oligomycin can be bound non-specifically.

2. The activity of oligomycin-inhibited processes in intact mitochondria and submitochondrial particles cannot be restored by treatment with egg lecithin or mitochondrial phospholipids.

3. Analysis of the kinetics of inhibition of State-3 respiration by oligomycin reveals that (i) after a certain lag period the inhibition by oligomycin is pseudo-first order with respect to the respiratory-control ratio, defined as the ratio of the respiratory rate at time t to that of the final inhibited site, (ii) the value of the pseudo-first-order rate constant (k₀) is dependent on the oligomycin: protein ratio, phospholipid: protein ratio, pH and temperature, (iii) the effects of various substrates and inhibitors of electron transfer on the kinetics of oligomycin inhibition can be explained by their effects on respiratory control.

4. A detailed model is proposed for the interaction of oligomycin with mitochondria. It is proposed that two conformations of the oligomycin-sensitive site are present, and that oligomycin specifically binds to the conformation that is involved in the induction of respiratory control.     

The number and localisation of adenine nucleotide-binding sites in beef-heart mitochondrial ATPase (F1) determined by photolabelling with 8-azido-ATP and 8-azido-ADP

1. When irradiated 8-azido-ATP becomes covalently bound (as the nitreno compound) to beef-heart mitochondrial ATPase (F₁) as the triphosphate, either in the absence or presence of Mg²⁺, label covalently bound is not hydrolysed.

2. In the presence of Mg²⁺ the nitreno-ATP is bound to both the and β subunits, mainly (63%) to the subunits.

3. After successive photolabelling of F₁ with 8-azido-ATP (no Mg²⁺) and 8-azido-ADP (with Mg²⁺) 4 mol label is bound to F₁, 2 mol to the and 2 mol to the β subunits.

4. When the order of photolabelling is reversed, much less 8-nitreno-ATP is bound to F₁ previously labelled with 8-nitreno-ADP. It is concluded that binding to the -subunits hinders binding to the β subunits.

5. F₁ that has been photolabelled with up to 4 mol label still contains 2 mol firmly bound adenine nucleotides per mol F₁.

6. It is concluded that at least 6 sites for adenine nucleotides are present in isolated F₁.     

The effects of phosphate and electron transport on the carbonyl cyanide m-chlorophenylhydrazone-induced ATPase of rat-liver mitochondria

1. The effects of phosphate and electron transport on the ATPase induced in ratliver mitochondria by the uncoupler carbonyl cyanide m-chlorophenylhydrazone have been measured at different uncoupler concentrations and compared with those of ATP, oligomycin and aurovertin.

2. The inhibitory action of respiratory-chain inhibitors on the ATPase activity, which is independent of the actual inhibitor used, is greatly delayed or prevented by the presence of uncoupler, and, in the case of rotenone, can be reversed completely by the subsequent addition of succinate (in the absence of uncoupler). These results can be explained on the basis of the proposal previously made by others that coupled electron transfer causes a structural change in the ATPase complex that results in a decreased affinity of the ATPase inhibitor for the mitochondrial ATPase.

3. Inorganic phosphate specifically stimulates the ATPase activity at high uncoupler concentrations (> 0.2 μM), but has no effect at low concentrations. The stimulation is prevented or abolished by sufficiently high concentrations of aurovertin.

4. Aurovertin prevents the inhibition of the uncoupler-induced ATPase by high uncoupler concentrations.

5. It is proposed that the steady-state concentration of endogenous P_i may be an important regulator of the turnover of the ATPase in intact mitochondria and that the inhibition of ATPase activity by high concentrations of uncoupler is at least partially mediated via changes in the concentration of endogenous P_i.     

Uncoupler-reversible inhibition of mitochondrial ATPase by metal chelates of bathophenanthroline. II. comparison with other inhibitors

(1) Trisbathophenanthroline-Fe²⁺(BPh₃Fe²⁺) alters the hyperbolic relationship between concentration of ATP and reaction velocity of F₁-ATPase to sigmoidal, with a simultaneous decrease in maximal velocity. (2) BPh₃Fe²⁺ binds to the β-subunit of F₁ and competes with the binding of aurovertin. The reversal of this effect by uncouplers in enhanced by ADP and diminished by ATP. BPh₃Fe²⁺ also changes the hyperbolic concentration dependence of aurovertin binding to sigmoidal. (3) BPh₃Fe²⁺ stabilizes F₁ against cold inactivation and cold dissociation in an uncoupler-reversible manner. (4) BPh₃Fe²⁺ efficiently protects F₁ against the light-induced inactivation occurring in the presence of Rose Bengal, and the effect is reversed by uncouplers. (5) The results are discussed in relation to the reaction mechanism of F₁-ATPase and other enzymes catalyzing the reversible hydrolysis of pyrophosphate bonds.     

Measurement of steady-state values of respiration rate and oxidation levels of respiratory pigments at low oxygen tensions. A new technique

1. An apparatus was developed for the simultaneous measurement of steady-state values of respiration rate and oxidation level of respiratory pigments at low oxygen tensions. An open reaction system is utilized. The liquid sample is in contact with a gas mixture whose oxygen tension can be increased linearly with time at a rate so slow that the system is always practically at a steady state.

2. Assuming Michaelis-Menten kinetics in the respiration, theoretical curves for oxygen tension in the liquid and oxidation level of the terminal oxidase during a linear increase of the oxygen tension in the gas were calculated.

3. Measurements were performed on rat liver mitochondria. Steady-state curves for oxygen tension in the liquid and oxidation level of the terminal oxidase, cytochrome a₃, obtained with coupled mitochondria resembled the theoretical curves. For uncoupled mitochondria the cytochrome a₃ curve was signmoidal, deviating strongly from the theoretical curve.

4. The apparent K_m for oxygen uptake of coupled mitochondria in the presence of pyruvate and malate, in the absence of phosphate was found to be 0.5 μM. In the case of uncoupled mitochondria the oxygen tension in the liquid could not be measured with sufficient accuracy to allow comparison with Michaelis-Menten kinetics. The apparent K_m for oxygen uptake was less than 0.05 μM.     

The phosphorylation potential generated by respiring mitochondria

1. 1. The phosphorylation potential, ΔG_P = ΔG₀′ + 1.36 log ([ATP]/[ADP][P_i]), where ΔG_O′ is the standard free energy of hydrolysis of ATP at a given pH, and [ATP], [ADP] and [P_i] refer to concentrations in the suspending medium, has been determined in rat-liver mitochondria under various conditions.

2. 2. The ATP/ADP ratio is relatively constant, over a 10-fold range of phosphate concentration. Thus, the phosphate potential is higher at low phosphate concentration. State-4 rat-liver mitochondria in the presence of succinate, oxygen and low concentrations of phosphate in State 4 maintain a phosphorylation potential of 16.1 kcal (67.3 kJ) per mole ATP.

3. 3. High concentrations of ATP inhibit ADP uptake, and it is suggested that this is the reason for the independence of the ATP/ADP ratio on the phosphate concentration. A steady-state ratio is set up dependent upon two processes that are relatively slow compared with State-3 respiration, namely ADP transport and ATP hydrolysis.

4. 4. The phosphorylation potential calculated from the concentrations of total ADP, ATP and P_i within State-4 mitochondria is 4.5 kcal/mole less than that in the suspending medium.

5. 5. It was shown experimentally that the phosphorylation potential cannot be calculated from the ΔG of the redox couple, the respiratory-control ratio and the P:O ratio, as has been suggested in the literature.

6. 6. The measured phosphorylation potential is 83% of that calculated from the span succinate to oxygen, assuming thermodynamic equilibrium, and 95% of that calculated from the span NADH to oxygen.

7. 7. Based on the measurements of the phosphorylation potential and of the redox potentials and redox states of redox components in mitochondria, ubiquinone and cytochrome b are found at their expected position at the junction of the phosphorylations at Sites 1 and 2. The iron-sulphur centres 2 and 5 and the iron-sulphur centre of succinate dehydrogenase also probably lie at this junction. Cytochrome a₃ lies at its expected junction between phosphorylation Sites 2 and 3. A number of electron carriers (cytochromes c, c₁, and a, the iron-sulphur centre of Complex III and the EPR-detectable copper), however, lie in the ‘no-man's land’ within Site 2.

8. 8. A phosphorylation potential of 16.1 kcal/mole corresponds to a membrane potential of 350 mV in State 4, on the basis of the chemiosmotic hypothesis.

Mitochondrial oxygen affinity as a function of redox and phosphate potenitals

1. The conditions under which mitochondria might catalyse a net reversal of oxidative phosphorylation are analysed.

2. Rat-liver mitochondria, incubated under such conditions, show a strongly diminished affinity for oxygen.

3. The velocity of respiration under these conditions is a hyperbolic function of the oxygen concentration.

4. The K_m for oxygen is less than 0.1 μM at low phosphate potential, irrespective of substrate, and 1–3 μM under reversal conditions.

5. The observed kinetics can be accounted for in a simple mechanism for cytochrome oxidase action.     

Purification and properties of ATPase inhibitor from rat liver mitochondria

(1) The ATPase inhibitor protein has been isolated from rat liver mitochondria in purified form. The molecular weight determined by sodium dodecyl sulfate gel electrophoresis is approximately 9500, and the isoelectric point is 8.9.

(2) The protein inhibits both the soluble ATPase and the particle-bound ATPase from rat liver mitochondria. It also inhibits ATPase activities of soluble F₁, and inhibitor-depleted submitochondrial particles derived from bovine heart mitochondria.

(3) On particle-bound ATPase the inhibitor has its maximal effect if incubated in the presence of Mg²⁺. ATP at slightly acidic pH.

(4) The inhibitor has a minimal effect on P_i-ATP exchange activity in sonicated submitochondrial particles. However, unexpectedly the inhibitor greatly stimulates P_i-ATP exchange activity in whole mitochondria while the low ATPase activity of the mitochondria is not affected. The possible mechanism of action of the inhibitor on intact mitochondria is offered.     

Studies on the mechanism of inhibition of the mitochondrial electron transport by antimycin. II. Antimycin as an allosteric inhibitor

1. Pretreatment of sub-mitochondrial particles with cholate results in a change in the curve describing inhibition by antimycin of the succinate-cytochrome c reductase from sigmoidal towards linear. This effect of cholate is reversed by partial removal of the cholate by dialysis, either in the absence or presence of antimycin.

2. Treatment with cholate has the same action on the sigmoidal effect curve of antimycin on the reducibility of cytochrome b. This is also reversed by dialysis.

3. The effect of antimycin on the displacement to the red of the -band of ferrocytochrome b, measured in the presence of succinate, NADH or reduced ubiquinone Q-2, is also described by a sigmoidal curve that is changed to a linear one by addition of cholate.

4. Linear displacement curves are obtained with menaquinol or Na₂S₂O₄.

5. It is proposed that antimycin is an allosteric inhibitor of the respiratory chain. This allosteric effect should be distinguished from the effect of antimycin on the “conformation stability” of Complex III.     

Azide inhibition of mitochondrial electron transport II. Spectral changes induced by azide

Azide inhibition of coupled mitochondrial transport is accompanied by spectral changes which indicate that the cytochrome a₃ is oxidized and cytochrome a reduced. The cytochrome a absorption band is shifted to shorter wavelengths in the azideinhibited system. This shift in the absorption band can be reversed by conditions leading to reduction of cytochrome a₃ such as uncouplers and anaerobiosis, or terminal inhibitors such as sulfide, cyanide or CO.

Titrations of the azide-induced spectral changes indicate the binding of one azide molecule in the complex, and that the dissociation constant is experimentally indistinguishable from the uncompetitive inhibitor constants for inhibition of State 3 respiration. The azide inhibition is postulated to involve the formation of a reduced cytochrome a azide compound which is unstable in the presence of reduced cytochrome a₃.     

Rhodamine 6G, inhibitor of both H-ejections from mitochondria energized with ATP and with respiratory substrates

Rhodamine 6G inhibited ATP hydrolysis by oligomycin-sensitive ATPase, purified from rat liver mitochondria, in good accord with the dose-response curve for its inhibition of energy transduction of ATP synthesis in mitochondria, but it did not inhibit ATP hydrolysis by purified F₁. Rhodamine 6G also inhibited both H⁺-ejections from mitochondria energized with respiratory substrates and with ATP.

The present findings show that the inhibitory effect of rhodamine 6G on energy transduction is not due to a modification of the transport system for adenine nucleotides, P_i, and respiratory substrates, and that the inhibition sites of rhodamine 6G are on components related with H⁺-ejection by redox components and also on F₀.     

On the nature of electron and energy transport in mitochondria. I. Multiple inhibition of mitochondrial respiration

1. A series of eight classical respiratory-chain inhibitors was studied. The slopes of State-3 respiratory rate versus dose plots are convex for antimycin, 2-n-heptyl-4-hydroxyquinoline-N-oxide (HOQNO), rotenone and sulfide, and concave for malonate, Amytal, cyanide and azide.

2. Plots of ADP: O ratio versus dose indicate uncoupling effects at higher concentrations of antimycin, HOQNO, cyanide and azide. On the other hand, sulfide and rotenone have no effect on the phosphorylating efficiency. Malonate increases the ADP: O ratio.

3. Two inhibitors can be combined in such a way that the total inhibition should be equal to the inhibition caused by the single inhibitors if each inhibitor affects respiration independently (additivity of inhibition). In practice, however, antagonism and synergism are also found.

4. Additivity of combined inhibition occurs where both inhibitors act on the same enzyme.

5. Antagonism is observed where the two inhibitors act on different enzymes of the same chain.

6. Synergism is found where the two inhibitors act on enzymes in different branches of a forked chain. This turns into normal additivity when the electron flow through both branches is made equal.

7. The results are compatible with the hypothesis that respiratory enzymes are arranged in chains. The possibility that the chains may be cross-linked or branched is discussed.     

Mitochondrial functions under hypoxic conditions: The steady states of cytochrome c reduction and of energy metabolism

1. The effect of low oxygen concentration on the oxidation-reduction states of cytochrome c and of pyridine nucleotide, on Ca²⁺ uptake, on the energy-linked reduction of pyridine nucleotide by succinate, and on the rate of oxygen consumption have been examined under various metabolic conditions, using pigeon heart mitochondria.

2. The oxygen concentration required to provide half-maximal reduction of cytochrome c (p50_c) ranges from 0.27 to 0.03 μM (0.2-0.02 Torr) depending upon the metabolic activity. There is a linear increase of the p50_c value with increasing respiratory rate.

3. The fraction of the normoxic respiration that is observed at p50_c is 70–90% under State 4 conditions, but is 30% under State 3 conditions.

4. The oxygen requirement for half-maximal reduction of pyridine nucleotide (p50_PN) varies less than p50_c, being 0.08 μM in State 3 and 0.06 μM in the uncoupled state.

5. The ability of the mitochondria to exhibit an energy-linked reduction of pyridine nucleotide by succinate disappears at an oxygen concentration of 0.09 μM (0.06 Torr). Below this oxygen concentration, endogenous Ca²⁺ begins to be released from the mitochondria. Thus, the critical oxygen concentration for bioenergetic function of mitochondria corresponds approximately to 50% reduction of pyridine nucleotide (p50_PN).     

Studies on yeast mitochondria: Some properties of mitochondria isolated from Saccharomyces Carlsbergensis grown in normal glucose medium

1. Mitochondria isolated from Saccharomyces Carlsbergensis are found to have three phosphorylation sites in the respiratory chain for the oxidation of NADH and NAD⁺-linked substrates and two for succinate oxidation. Freshly isolated mitochondria exist in an inhibited state with no respiratory control, but on ageing for 2–3 h a good coupled state is obtained. -Ketogultarate and -glycerophosphate are poorly oxidized in these mitochondria.

2. Exogenous NADH is a very good substrate for yeast mitochondrial respiration and apparently has a very low K_m. However, one-third of the added NADH is not available for oxidation probably due to some form of compartmentation. Studies of both oxygen uptake and the redox changes of cytochrome b show complete oxidation of two-third of the added NADH.

3. Difference spectra of yeast mitochondria at liquid-nitrogen temperatures show all the characteristic peaks of cytochromes a (600 nm), b (558, 525 and 428 nm), c₁ (552 nm) and c (545 and 516 nm).

4. The reduction of cytochrome b by dicumarol in antimycin A inhibited mitochondria provides evidence for an energy conservation site on the substrate side of cytochrome b.

5. In the absence of added ADP, the oxidation of malate and pyruvate occurs in the yeast mitochondria in a new respiratory state (State X) where the oxygen uptake occurs at State 4 rate but the redox level of the flavins, cytochrome b and c are similar to State 3. State X respiration is believed to be due to depletion of the high energy intermediate C I caused by the substrate anions accumulation.

6. The responses of yeast mitochondria to Ca²⁺ are qualitatively similar to those in rat liver mitochondria, particularly with respect to respiratory stimulation, membrane alkalinization and its accumulation in the mitochondria with succinate as the substrate in the presence and absence of acetate.     

Adenine nucleotide translocation in yeast mitochondria. Effect of inhibitors of mitochondrial biogenesis on the ADP translocase

1. Optimal test conditions for adenine nucleotide translocation in Candida utilis mitochondria are a standard medium, consisting of 0.63 M mannitol, 2 mM EDTA (or ethylene glycol tetraacetic acid, EGTA), 10 mM morpholinopropane sulfonic acid (pH 6.8), and a temperature of 0 °C.

2. Adenine nucleotide translocation in C. utilis mitochondria is an exchange-diffusion process. The whole pool of internal adenine nucleotides is exchangeable, ADP being the most readily exchangeable nucleotide. The rate of mitochondrial ADP exchange, but not the K_m value, depends on growth conditions. At 0 °C, the rate is about 3 to 4 nmoles ADP/min per mg protein for mitochondria obtained from yeast grown in the presence of 1.5% glucose; it rises to 11.5 nmoles when glucose is replaced by 3% ethanol in the growth medium. The K_m value for ADP is 2 μM. The Q₁₀ is about 2 between 0 and 20 °C. Among other exchangeable adenine nucleotides are ATP, dADP and the methylene and the hypophosphate analogues of ADP. Unlike mammalian mitochondria, C. utilis mitochondria are able to transport external UDP by a carboxyatractyloside-sensitive process.

3. Under conditions of oxidative phosphorylation (phosphate and substrate present in an aerated medium), added ADP is exchanged with internal ATP. A higher ATP/ADP ratio was found in the extramitochondrial space than in the intramito-chondrial space. The difference between the calculated phosphate potentials in the two spaces was 0.9–1.7 kcal/mole.

4. Atractyloside, carboxyatractyloside, bongkrekic acid and palmityl-CoA inhibit mitochondrial adenine nucleotide translocation in C. utilis as they do in mammalian mitochondria, but 2 to 4 times less efficiently. The inhibition due to atractyloside or palmityl-CoA is competitive with respect to ADP whereas that due to bongkrekic acid and carboxyatractyloside is non-competitive. Carboxyatractyloside and atractyloside inhibitions are additive. The apparent K_d for the binding of [³⁵S]-carboxyatractyloside and [¹⁴C]bongkrekic acid is 10–15 nM and the concentration of sites 0.4–0.6 nmole/mg protein in both cases. [³⁵S]Carboxyatractyloside binding is competitively displaced by atractyloside and vice versa.

5. Binding of [¹⁴C]ADP has been carried out with mitochondria depleted of their endogenous adenine nucleotides by incubation with phosphate and Mg²⁺ at 20 °C. The amount of bound [¹⁴C]ADP which is atractyloside removable is 0.08–0.16 nmole/mg protein.

6. The rate of ADP transport is quite different in mitochondria isolated from C. utilis, according to whether it is grown on glucose, or on ethanol or in the presence of chloramphenicol; for instance, it decreases by 10 times when 3% ethanol in the growth medium is replaced by 10% glucose and by 5 times when chloramphenicol is added to the medium. These variations are accompanied by parallel variations in cytochrome aa₃. The number of atractyloside-sensitive ADP binding sites is not modified by the above conditions of culture, nor the number of [³⁵S]carboxyatractyloside binding sites. The affinity for ADP is apparently not significantly modified, nor the size of the endogenous adenine nucleotide pool. In contrast to glucose repression or chloramphenicol inhibition, semi-anaerobiosis in C. utilis lowers significantly the mitochondrial binding capacity for carboxyatractyloside. Strict anaerobiosis in S. cerevisiae results in a practical loss of the cytochrome oxidase activity, and also of the carboxyatractyloside and ADP binding capacity. Transition from anaerobiosis to aerobiosis restores the cytochrome oxidase activity and the ADP and carboxyatractyloside binding capacities.     

Effect of filipin on Rat-liver and yeast mitochondria

1. Under the appropriate conditions intact yeast and mammalian mitochondria exhibit a heretofore unobserved sensitivity to the polyene antibiotic, filipin. The activity of the “filipin complex” (Filipins I, II, III and IV) is shown to be primarily due to the component designated Filipin II.

2. Yeast mitochondria treated with filipin complex, or purified Filipin II, exhibit “uncoupled” succinate oxidation and inhibited -ketoglutarate oxidation. Maximum filipin effect is observed at a concentration of 4 mM Filipin II. Rat-liver mitochondria are more sensitive to filipin than yeast mitochondria, and respiratory inhibition is observed regardless of substrate.

3. In liver mitochondria filipin-inhibited respiration is not relieved by Mg²⁺, K⁺, Ca²⁺ or 2,4-dinitrophenol, but is reversed by cytochrome c.

4. It is proposed that filipin treatment leads to altered membrane permeability and that respiratory inhibition is due to a loss of endogenous respiratory cofactors or an inactivation of primary dehydrogenases. The filipin-uncoupled yeast respiration may likewise be attributed to an altered phosphate permeability of the yeast mitochondrial membranes.     

Phosphate transport in rat-liver mitochondria

1. The kinetics of the efflux of P_i and malate as well as the relationship between P_i transport and intra- and extramitochondrial pH changes were studied in rat-liver mitochondria in the presence of rotenone and oligomycin at different pH's.

2. At high pH a fast efflux of P_i from the mitochondria occurs in the first few seconds, followed by a slow re-entry of P_i into the mitochondria. Under the same conditions the exit of malate shows a time lag of 2–4 sec. The exit of malate coincides with the re-entry of P_i.

3. In the presence of butylmalonate the exit of endogenous P_i is coupled with a concomitant alkalinization of the mitochondrial matrix space, as calculated from the distribution of 5,5-[¹⁴C]dimethyloxazolidine-2,4-dione.

4. The stoicheiometry of the P_i-hydroxyl exchange was found to be 1:1.

5. The kinetics of P_i transport are consistent with previous observations that there is a direct exchange between OH⁻ and P_i, but not between OH⁻ and malate. The equilibrium distribution of H₂PO₄⁻ and OH⁻ deviates from the Donnan distribution. This may be explained by assuming a pH-dependent binding of P_i in the mitochondria.     

Characterization of a mutant of Schizosaccharomyces pombe lacking cytochrome b-566

1. A chromosomal respiration-deficient mutant of the petite-negative yeast Schizosaccharomyces pombe was isolated. Its mitochondria show respiration rates of about 7% of the wild-type respiration with NADH and succinate as substrate, and 45% with ascorbate in the presence of tetramethyl-p-phenylenediamine. Oxidation of NADH and succinate is insensitive to antimycin and cyanide and that of ascorbate is much less sensitive to cyanide than the wild type.

2. The amounts of cytochromes c₁ and aa₃ are similar in the mutant and wild type. Cytochrome b-566 could not be detected in low-temperature spectra after reduction with various substrates or dithionite. A b-558 is, however, present.

3. The b-cytochromes in the mutant are not reduced by NADH or succinate during the steady state even after addition of ubiquinone-1. QH₂-3: cytochrome c reductase activity is very low and succinate oxidation is highly stimulated by phenazine methosulphate.

4. Antimycin does not bind to either oxidized or reduced mitochondrial particles of the mutant.

5. In contrast to the b-cytochromes of the wild type, b-558 in the mutant reacts with CO.

6. Cytochromes aa₃, c and c₁ are partly reduced in aerated submitochondrial particles isolated from the mutant and the EPR signal of Cu (II), measured at 35°K, is detectable only after the addition of ferricyanide. In the mutant, a signal with a trough at g = 2.01 is found, in addition to the signal at g = 1.98 found in the wild type.

7. The ATPase activity of particles isolated from the mutant is much lower than in the wild type but is still inhibited by oligomycin.     

The F1-ATPase beta-subunit is the putative enterostatin receptor

It has been suggested that the F₁-ATPase β-subunit is the enterostatin receptor. We investigated the binding activity of the purified protein with a labeled antagonist, β-casomorphin_1–7, in the absence and presence of cold enterostatin. ¹²⁵I-β-casomorphin_1–7 weakly binds to the rat F₁-ATPase β-subunit. Binding was promoted by low concentrations of cold enterostatin but displaced by higher concentrations. To study the relationship between binding activity and feeding behavior, we examined the ability of a number of enterostatin analogs to affect β-casomorphin_1–7 binding to the F₁-ATPase β-subunit. Peptides that suppressed food intake promoted β-casomorphin_1–7 binding whereas peptides that stimulated food intake or did not affect the food intake displaced β-casomorphin_1–7 binding. Surface plasmon resonance measurements show that the β-subunit of F₁-ATPase binds immobilized enterostatin with a dissociation constant of 150 nM, where no binding could be detected for the assembled F₁-ATPase complex. Western blot analysis showed the F₁-ATPase β-subunit was present on plasma and mitochondrial membranes of rat liver and amygdala. The data provides evidence that the F₁-ATPase β-subunit is the enterostatin receptor and suggests that enterostatin and β-casomorphin_1–7 bind to distinct sites on the protein.